The main goal of this proposal is to characterize the number of protonic sites and their mode of participation in the catalysis of substrate hydrolysis by hemostatic and thrombolytic enzymes. The target enzymes are human thrombin, human factor Xa, activated protein C (APC) and bovine plasmin, some of the most sophisticated members of the serine protease family. Characterization of full and partial solvent isotope effects will be carried out for the enzyme-catalyzed hydrolysis of sets of three types of substrates: 1) Chromogenic or fluorogenic di- to pentapeptide amide substrates will test the effect of P1-P5 residues. 2) To test the collective contribution of P1-P5 and about '-P5' sites, but without exosites, fluorescence-quenched substrates with an N-terminal 2-aminobenzoyl (AB)-Val fluorophore and a C-terminal Lys-2, 4-dinitrophenyl (DNP) quencher and an Asp-OH to enhance solubility, will be studied with thrombin. 3) The exosite dependence of the effect of the N-acyl, or leaving group binding site on the extent of protonic participation will be evaluated from studies of selected natural reactions. Naturally occurring substrates of thrombin achieve specific binding to the enzyme at designated exosites remote from the active site. Preliminary experiments will be conducted to find the optimal conditions for the measurements of Michaelis Menten parameters and then their dependence on pH will be studied in H2O and D20. Specific kinetic parameters (kcat and kcat /Km) obtained in 8 to 10 mixtures of isotopic waters each at n, a particular atom fraction of D in the medium, will be fit to models derived from the Gross-Butlet equation. Fractionation factors for multiproton transfer at the transition state (s) and for solvent contribution will be calculated. The statistically most significant model will be sought using the ?2 and F-tests. The results of these studies will promote the basic understanding of enzyme catalytic power and enzyme evolutionary theory, and also provide guidelines to the development of transition state analog inhibitors in targeted drug design.